Method for measuring aromatase activity

ABSTRACT

The present invention relates to compounds useful for measuring aromatase activity. The invention further provides methods for measuring aromatase activity and for screening test agents which modulate aromatase activity. A kit is also provided for use in such screening methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/561,817 filed Dec. 7, 2006, which is a filing under 35 U.S.C. §371and claims priority to international patent application numberPCT/GB2004/003341 filed Jul. 30, 2004, published on Feb. 10, 2005 as WO2005/012901, which claims priority to application number 0317743.3 filedin Great Britain on Jul. 30, 2003.

FIELD OF THE INVENTION

The present invention relates to compounds, methods and a kit formeasuring aromatase activity. The invention can be used for determiningboth in vitro and in vivo enzyme activity and foridentifying/characterising inhibitors.

BACKGROUND OF THE INVENTION

Assays for measuring enzyme activity are widely employed in thepharmaceutical and environmental sciences. With the advent ofcombinatorial chemistry and high throughput screening there is a growingneed for simple, sensitive and cost-effective assays to screen forpotential modulators of enzyme activity.

The enzyme aromatase cytochrome P450 19 A1 (EC 1.14.14.1) is the productof the CYP19 gene, a member of the P450 superfamily of genes. Aromatasecatalyses the rate-limiting step in oestrogen biosynthesis, theconversion of C₁₉ androgenic steroids to the corresponding oestrogen(FIG. 1), a reaction termed aromatisation since it converts the Δ⁴-3-onering of the androgen to the phenolic A-ring of oestrogen (Ciolino etal., 2000, British Journal of Cancer, 83, 333-337).

Oestrogens are the most important etiological factors in the growth anddevelopment of many breast carcinomas in both pre- and post-menopausalwomen. Breast tumours from post-menopausal women contain high levels of17β-oestradiol despite the presence of low plasma 17β-oestradiolconcentrations. It is now widely accepted that breast tumours cansynthesise 17β-oestradiol from adrenal androgen precursors. Synthesisoccurs through the aromitisation of androstenedione to oestrone byaromatase, followed by conversion of oestrone to 17β-oestradiol by17β-hydroxysteroid dehydrogenase type 1 (James et al., 2001,Endocrinology 142, 1497-1505).

When measured in-vitro, aromatase activity was found to be higher inbreast tumours than in adjacent or healthy fat cells. Furthermore,adipose stromal cells surrounding cancerous cells have been shown tocontain higher levels of aromatase mRNA than corresponding cells innon-cancerous areas (Chen et al., 1999, Endocrine-Related Cancer, 6,149-156). Thus aromatase activity in tumours or surrounding tissue isbelieved to play a significant role in promoting tumour growth due tolocal production of oestrogen.

Aromatase offers a key point of intervention in the treatment of breastcancer by reducing the activity and consequently the level of oestrogensynthesised at the site of the tumour. Thus aromatase inhibitors providesignificant benefit to many breast cancer patients (James et al., 2001,Endocrinology 142, 1497-1505).

Aromatase is an important enzyme not only from a medical andpharmaceutical viewpoint in the treatment of breast cancer but also froman environmental perspective because inhibitors have been identified aspotential environmental toxins, or so called ‘endocrine disrupters’ (Maket al., 1999, Environmental Health Perspectives, 107, 855-860). Thedevelopment of a simple, high throughput screening assay to identifymodulators and particularly inhibitors of aromatase activity is thus ofconsiderable commercial interest.

Fluorescence Detection Methods

Fluorescence-based assays offer significant advantages overradiochemical, ELISA, antibody and more traditional techniques formeasuring enzyme activity in terms of simplicity of handling,sensitivity, cost and ease of automation. Recently there has beenconsiderable interest in the application of fluorescence resonanceenergy transfer (FRET) assays which involve the use of substrates havingdonor and quenching acceptors on the same molecule. WO 94/28166, forexample, reports the use of such FRET labels attached to a polypeptidesubstrate which fluoresce more intensely on hydrolysis by a protease.

While FRET techniques offer greater sensitivity and reliability for usein screening assays than simple fluorescent intensity techniques, thesubstrates are considerably more expensive to prepare and purify due totheir complex nature. Thus the preparation of FRET labels is demandingin terms of both analytical and/or purification and material costs.Furthermore the only method for distinguishing conventional fluorescentor FRET labels is by their absorption and emission spectra.

Fluorescence lifetime measurements that may be utilised in the presentinvention offer significant advantages over conventional fluorescencetechniques that are based solely on quantifying fluorescence intensity.Fluorescence lifetime is determined from the same spectrally resolvedintensity signal, but is additionally resolved in the temporal domain.Fluorescence lifetime techniques provide greater discrimination becausethe signal is largely unaffected by ‘background noise’. A furtheradvantage with this technique is that several different events can bemeasured simultaneously by selecting labels having distinguishablelifetimes, thus enabling multiplexing. In addition, measurements offluorescence lifetime are unaffected by concentration effects andphotobleaching.

Aromatase Assays

Several assay formats have been reported for the measurement ofaromatase activity. These can be divided into two categories dependingon the use of a ‘natural’ or a surrogate substrate. Detectionmethodologies have included the use of radioisotopic tracers (e.g.Thompson & Siiteri, 1974, Journal of Biological Chemistry, 249,5364-5372), fluorescence intensity (Crespi et al., AnalyticalBiochemistry, 1997, 248, 188-190), enzyme activity (e.g. Chabab et al.,1986, Journal of Steroid Biochemistry, 25, 165-169) and fast liquidchromatography (Fauss & Pyerin, 1993, Analytical Biochemistry, 210,421-423).

Odum and Ashby (Toxicology Letters (2002), 129, 119-122) describe aradiometric assay for measuring aromatase activity using the ‘tritiatedwater assay’. The assay quantifies enzyme activity based on the releaseof ³H as ³H₂O from the 1β position of the substrate duringaromatisation. A final reaction contained rat ovary microsomes and anNADPH generating system together with the substrate1β(³H)-androstenedione and potential aromatase inhibitors in dimethylsulphoxide. Reactions were started by addition of the substrate and werecarried out at 37° C. for 30 min. Reactions were stopped by addition ofchloroform-methanol and the mixture shaken for 60 s. After removal ofthe solvent, a suspension of dextran-coated charcoal was added. Themixture was left for 1 h at 4° C., centrifuged and 500 μl of thesupernatant added to scintillant and counted in a liquid scintillationcounter.

Although this assay has been widely used in the literature (e.g. WO03/045925) as a means for identifying potential inhibitors it is clearlynot amenable to high throughput procedures as it is a labour intensiveand time-consuming, requiring radiolabelled substrate.

Crespi et al. (Analytical Biochemistry (1997), 248, 188-190) describe amicrotitre plate-based fluorimetric intensity assay that can be used tomeasure the activity of recombinant human aromatase expressed in insectcells and prepared as microsomes. The assay uses dibenzylfluorescein(DBF) as the substrate and reports a number of IC5₅₀ values that are inmany cases different from reported values. These differences arereportedly due to variation in methodology such as substrate choice andthe use of cell based systems. The use of a ‘surrogate’ substrate inthis second format may explain why the IC5₅₀ differ from the publishedvalues.

There is therefore a continued need in the pharmaceutical andenvironmental sciences for improved fluorescence-based assays formeasuring aromatase activity. Such assays may have one or more of thefollowing attributes: homogeneity, high sensitivity, good reliability,robustness, simplicity of use, low cost, ease of automation, labelspecificity and/or more than one form of detection for distinguishinglabelled compounds. Preferably the improved assays display more than oneof these features and preferably they display all of these features. Thepresent invention seeks to provide novel reagents and methods forperforming such an assay.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention, there is provideda compound of Formula I:

R-L-S   (I)

wherein R is a fluorescent dye molecule;

-   -   L is an optional linkage group containing one or more atoms        comprising hydrocarbon chains which may also contain other atoms        such as N, O and S; and    -   S is a molecule comprising a substrate group of the enzyme        aromatase        characterised in that the fluorescence signal of said compound        changes in respect of fluorescence intensity or fluorescence        lifetime when the compound is acted upon by an enzyme with        aromatase activity.

Suitably, R is selected from the group consisting of fluorescein,rhodamine, coumarin, BODIPY™ dye, phenoxazine, cyanine, Alexa FLUOR®,merocyanine, CY™3B, CY™5, CY™5.5, CY™7, acridone, quinacridone andsquarate dyes.

A range of fluorescent labels are commercially available which could beused as a fluorescent reporter moiety R in accordance with the presentinvention. Examples include, but are not limited to, oxazine (e.g. MR121, JA 242, JA 243) and rhodamine derivatives (e.g. JA 165, JA 167, JA169) as described in WO 02/081509. Other examples (as described in WO02/056670) include, but are not limited to CY™5, CY™5.5 and CY™7 (GEHealthcare); merocyanine (Few Chemicals), IRD41 and IRD700 (Licor);NIR-1 and IC5-OSu (Dojindo); Alexa FLUOR® 660 & Alexa FLUOR® 680(Molecular Probes); LaJolla Blue (Diatron); FAR-Blue, FAR-Green One &FAR-Green Two (Innosense); ADS 790-NS and ADS 821-NS (American DyeSource); indocyanine green (ICG) and its analogues (U.S. Pat. No.5,968,479); indotricarbocyanine (ITC, WO 98/47538); fluorescent quantumdots (zinc sulfide-capped cadimium selenide nanocrystals—QuantumDotCorp.) and chelated lanthanide compounds (fluorescent lanthanide metalsinclude europium and terbium).

Preferably, R is an acridone dye, as described in WO 02/099424, offormula II:

wherein:

-   groups R² and R³ are attached to the Z¹ ring structure and groups R⁴    and R⁵ are attached to the Z² ring structure;-   Z¹ and Z² independently represent the atoms necessary to complete    one or two fused ring aromatic or heteroaromatic systems, each ring    having five or six atoms selected from carbon atoms and optionally    no more than two atoms selected from oxygen, nitrogen and sulphur;-   R¹, R², R³, R⁴ and R⁵ are independently selected from hydrogen,    halogen, amide, hydroxyl, cyano, amino, mono- or di-C₁-C₄    alkyl-substituted amino, sulphydryl, carbonyl, C₁-C₆ alkoxy, aryl,    heteroaryl, C₁-C₂₀ alkyl, aralkyl, the group -E-F where E is a    spacer group having a chain from 1-60 atoms selected from the group    consisting of carbon, nitrogen, oxygen, sulphur and phosphorus atoms    and F is a target bonding group; and the group —(CH₂—)_(n)Y where Y    is selected from sulphonate, sulphate, phosphonate, phosphate,    quaternary ammonium and carboxyl and n is zero or an integer from 1    to 6.

Suitably, the target bonding group F is a reactive or functional group.A reactive group of the fluorescent dyes according to formula (II) canreact under suitable conditions with a functional group of the substrate(i.e. group L or X); a functional group of a compound according toformula (II) can react under suitable conditions with a reactive groupof the substrate. By virtue of these reactive and functional groups, thefluorescent dyes according to formula (II) may be reacted with andcovalently bond to the substrate, such that the substrate becomeslabelled with the fluorescent dye.

Preferably, when F is a reactive group, it is selected from the groupconsisting of succinimidyl ester, sulpho-succinimidyl ester,isothiocyanate, maleimide, haloacetamide, acid halide, vinylsulphone,dichlorotriazine, carbodiimide, hydrazide and phosphoramidite.Preferably, when F is a functional group, it is selected from hydroxy,amino, sulphydryl, imidazole, carbonyl including aldehyde and ketone,phosphate and thiophosphate.

Preferably, R is a quinacridone dye, as described in WO 02/099432, ofFormula III:

wherein:

-   groups R³ and R⁴ are attached to the Z¹ ring structure and groups R⁵    and R⁶ are attached to the Z² ring structure;-   Z¹ and Z² independently represent the atoms necessary to complete    one or two fused ring aromatic or heteroaromatic systems, each ring    having five or six atoms selected from carbon atoms and optionally    no more than two atoms selected from oxygen, nitrogen and sulphur;-   R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are independently selected from    hydrogen, halogen, amide, hydroxyl, cyano, amino, mono- or di-C₁-C₄    alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C₁-C₆    alkoxy, aryl, heteroaryl, C₁-C₂₀ alkyl, aralkyl, the group -E-F    where E is a spacer group having a chain from 1-60 atoms selected    from the group consisting of carbon, nitrogen, oxygen, sulphur and    phosphorus atoms and F is a target bonding group; and the group    —(CH₂—)_(n)Y where Y is selected from sulphonate, sulphate,    phosphonate, phosphate, quaternary ammonium and carboxyl and n is    zero or an integer from 1 to 6.

Suitably, the target bonding group F is a reactive or functional group.A reactive group of the fluorescent dyes according to formula (III) canreact under suitable conditions with a functional group of thesubstrate; a functional group of a compound according to formula (III)can react under suitable conditions with a reactive group of thesubstrate. By virtue of these reactive and functional groups, thefluorescent dyes according to formula (III) may be reacted with andcovalently bond to the substrate, such that the substrate becomeslabelled with the fluorescent dye.

Preferably, when F is a reactive group, it is selected from the groupconsisting of succinimidyl ester, sulpho-succinimidyl ester,isothiocyanate, maleimide, haloacetamide, acid halide, vinylsulphone,dichlorotriazine, carbodiimide, hydrazide and phosphoramidite.Preferably, when F is a functional group, it is selected from hydroxy,amino, sulphydryl, imidazole, carbonyl including aldehyde and ketone,phosphate and thiophosphate.

Preferred examples of acridone and quinacridone dyes (and theircorresponding lifetimes (nano seconds)) are shown as compounds (IV),(V), (VI), (VII) and (VIII) in Table 1 as their NHS(N-hydroxysuccinimidyl)esters:

TABLE 1

Suitably, L is a linker group containing from 1 to 40 linked atomsselected from carbon atoms which may optionally include one or moregroups selected from —NR′—, —O—, —S—, —CH═CH—, —C≡C—, —CONH— andphenylenyl groups, wherein R′ is selected from hydrogen and C1 to C4alkyl.

Suitably, L is a linker group containing from 2 to 30 atoms, preferablyfrom 6 to 20 atoms.

Preferably, L is a linker group selected from the group:

-   {(—CHR′—)p-Q-(—CHR′—)r}s where each Q is selected from CHR′, NR′, O,    —CH═CH—, Ar and —CONH—; each R′ is independently hydrogen or C1 to    C4 alkyl; each p is independently 0 to 5; each r is independently 0    to 5; and s is either 1 or 2. More preferably, Q is selected from    the group consisting of —CHR′—, —O— and —CONH—, where R′ is hydrogen    or C1 to C4 alkyl.

Preferably, Group S is a steroid of Formula IX or a derivative thereof

wherein:

-   R¹ and R² are selected from H and methyl;-   R³ is selected from H, C₁-C₈ alkyl, cyano, —(CH₂)_(k)—OR^(a);    —(CH₂)_(k)—COOR^(a); —(CH₂)_(k)—SO₃R^(a); —(CH₂)_(k)—CHO,    —(CH₂)_(k)—NR^(b)R^(c) and —(CH₂)_(k)—COR^(d);-   R⁴ is selected from H, —COR^(a) and hydroxyl;-   R⁵ is selected from H, —COR^(a), hydroxyl, cyano and halide;-   R⁶ is selected from H and hydroxyl;-   R⁷, R⁸ and R⁹ are independently selected from H, —COR^(a) and    hydroxyl;-   R¹⁰ is selected from H and halide; and-   where R^(a) is selected from H and C1-C4 alkyl, optionally    substituted with OH; R^(b) and R^(c) are selected from H and C₁-C₄    alkyl;-   R^(d) is selected from C₁-C₈ alkyl or C₁-C₈ alkyl optionally    substituted with COOR^(a), OH, OR^(a) or SO₃R^(a);    and k is zero or an integer from 1 to 8.

Halogen and halide groups are selected from fluorine, chlorine, bromineand iodine.

Suitably, Group S is a steroid selected from the group of steroidfamilies consisting of 4-androsten-3-one, 4-cholesten-3-one,4-estren-3-one and 4-pregnen-3-one derivatives.

Preferably, Group S is androstenedione of Formula X or a derivativethereof

Preferably, Group S is testosterone of Formula XI or a derivativethereof

In a preferred embodiment of the first aspect, there is provided acompound of Formula XII

In a second aspect of the present invention, there is provided a methodfor measuring aromatase activity in a sample, the method comprising thesteps of:

-   i) measuring the fluorescence intensity or fluorescence lifetime of    a compound according to any preceding claim prior to adding it to    said sample;-   ii) adding said compound to said sample under conditions which    favour aromatase activity, and-   iii) measuring a change in fluorescence intensity or fluorescence    lifetime of said compound following step ii);

wherein said change in fluorescence intensity or fluorescence lifetimecan be used to determine aromatase activity.

Suitably, the sample is selected from the group consisting of extract,cell, tissue and organism. The cell or organism may be naturallyoccurring or may be a recombinant cell or organism which has beengenetically engineered to over-express a particular protein, such asaromatase.

In a third aspect of the present invention, there is provided a methodof screening for a test agent whose effect upon the activity ofaromatase is to be determined, said method comprising the steps of:

-   i) performing the method as hereinbefore described in the presence    of said agent; and-   ii) comparing the activity of said aromatase in the presence of the    agent with a known value for the activity of aromatase in the    absence of the agent;    wherein a difference between the activity of the aromatase in the    presence of the agent and said known value in the absence of the    agent is indicative of the effect of the test agent upon the    activity of aromatase.

A test agent may be, for example, any organic or inorganic compound suchas a synthetic molecule or a natural product (e.g. peptide,oligonucleotide), or may be an energy form (e.g. light or heat or otherforms of electro magnetic radiation).

Suitably, the known value is stored upon an electronic database.Optionally, the value may be normalised (for example, to represent 100%aromatase activity) and compared to the normalised activity of theenzyme in the presence of the test agent. In this way, only test agentsaffecting enzyme activity by a certain minimum amount may be selectedfor further evaluation.

According to fourth aspect of the present invention, there is provided amethod of screening for a test agent whose effect upon the activity ofaromatase is to be determined, said method comprising the steps of:

-   i) performing the method of measuring aromatase activity as    hereinbefore described in the presence and in the absence of the    agent; and-   ii) determining the activity of said enzyme in the presence and in    the absence of the agent;    wherein a difference between the activity of aromatase in the    presence and in the absence of the agent is indicative of the effect    of the test agent upon the activity of aromatase.

Suitably, the difference in activity between the activity of the enzymein the absence and in the presence of the agent is normalised, storedelectronically and compared with a value of a reference compound. Thus,for example, the difference in activity may be stored as a percentageinhibition (or percentage stimulation) on an electronic database andthis value compared to the corresponding value for a standard inhibitorof aromatase. In this way, only test agents meeting a certainpre-determined threshold (e.g. as being as effective or more effectivethan the reference compound) may be selected as being of interest forfurther testing.

The assay method according to the present invention is preferablyperformed in the wells of a multiwell plate, e.g. a microtitre platehaving 24, 96, 384 or higher densities of wells eg. 864 or 1536 wells.Alternatively, the assay may be conducted in assay tubes or in themicrochannels of a multifluidic device or in a FACS machine. In atypical assay, a sample containing the substance of interest is mixedwith the reaction mixture in a well. The reaction is initiated by theaddition of enzyme. The reaction is allowed to proceed for a fixed timeand stopped with a stop reagent (for example, EDTA).

The reaction mixture can be pre-dispensed into the wells of such aplate.

Typically, enzyme assays are performed under “stopped” conditions. Bythis it is meant that the reaction is allowed to proceed for apredetermined period and then terminated with a stop reagent. The natureof the stop reagent is typically a strong inhibitor of the enzyme and isoften non-specific, for example, EDTA, is used to sequester metal ionsthat are normally present for enzyme activity. In embodiments of thesecond, third and fourth aspects, assays for aromatase activity eitherin the presence of or in the absence on a test compound, may beperformed under continuous measurement. Because the fluorescenceintensity and/or lifetime of the labelled substrate is monitoredcontinuously and can be seen to change continuously, the labelledsubstrate does not need separation from the product of the enzymaticreaction. A time-course of the reaction may be obtained in this manner,thus allowing kinetic studies to be performed in real time.

In general the assay will consist of several components, typically theenzyme, substrate, cofactors, metal ions, buffer salts and possibly testor standard inhibitor compounds.

Additionally it may be necessary to run the assays in the presence oflow percentages of organic solvents such as DMSO. In this invention itis possible to add any of the reagents to the mix whilst omitting acritical component in any order. This type of reaction can then bemonitored for non-specific effects. It is also possible to constructmixture with no enzyme for further controls. Due to the nature of thereactions, it is then possible to add the final component and monitorchanges either in real time or by stopping the reaction at some point inthe future.

The methods of the invention can be carried out in samples derived fromcells, tissues, organisms and extracts. Biological samples may, forexample, be homogenates, lysates or extracts prepared from wholeorganisms, parts of an organism or tissues. For example, the assay canbe conducted on a variety of body fluids such as blood, mucus, lymphaticfluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid,urine, vaginal fluid and semen. In particular, the assay may beconducted on adipose or breast tissues and cells.

Furthermore, it is possible to conduct the assay in media, such asnutrient broth or similar media, where it is possible to grow eithereukaroytic or prokaryotic cells. Cells engineered to over-expressaromatase, such as JEG3 choriocarcinoma cells obtained from ATCC(Bhatnager et al., 2001, Journal of Steroid Biochemistry and MolecularBiology, 76, 199-202) are particularly useful for screening inhibitors.

Suitably, conventional detection methods can be employed to measurefluorescence intensity and/or the lifetime of the label. These methodsinclude instruments using photo-multiplier tubes as detection devices.Several approaches are possible using these methods; e.g.

-   i) methods based upon time correlated single photon counting (cf.    Principles of

Fluorescence Spectroscopy, (Chapter4) ed. J R Lakowicz, Second Edition,1999, Kluwer/Academic Press)

-   ii) methods based upon frequency domain/phase modulation (cf.    Principles of Fluorescence Spectroscopy, (Chapter5) ed. J R    Lakowicz, Second Edition, 1999, Kluwer/Academic Press)-   iii) methods based upon time gating (cf. Sanders et al., (1995)    Analytical Biochemistry, 227 (2), 302-308).

Measurement of fluorescent intensity may be performed by means of acharge coupled device (CCD) imager, such as a scanning imager or an areaimager, to image all of the wells of a multiwell plate. The LEADSEEKER™(GE Healthcare) system features a CCD camera allowing imaging of highdensity microtitre plates in a single pass. Imaging is quantitative andrapid, and instrumentation suitable for imaging applications can nowsimultaneously image the whole of a multiwell plate.

According to a fifth aspect of the present invention, there is provideda method for measuring the distribution of a compound as hereinbeforedescribed within a tissue, wherein the compound is capable of beingtaken up by a living cell within the tissue, the method comprising thesteps of:

-   i) measuring the fluorescence intensity or fluorescence lifetime of    the compound in a cell-free environment or a parental host cell;-   ii) adding the compound to one or more cells or a cell engineered to    over-express aromatase, and-   iii) measuring the fluorescence intensity or lifetime of the    compound following step ii);    wherein a change in fluorescence intensity or fluorescence lifetime    indicates aromatase activity and can be used to determine the    distribution of the compound. It will be understood that cells which    have been genetically engineered to over-express aromatase compared    to their parental host cells will exhibit significantly higher    levels of enzyme activity.

Suitably, the distribution of the compound within the tissue of a firstsubject is compared with the distribution of the compound within thetissue of a second subject.

Suitably, the subject is selected from the group consisting of mammal,plant, insect, fish, bird, fly, nematode and algae. Preferably, themammal is a mouse or a rat.

In a sixth aspect of the present invention, there is provided the use ofa compound as hereinbefore described for measuring aromatase activity asan in vitro or an in vivo imaging probe.

In a seventh aspect of the present invention, there is provided a methodof diagnosing a disease caused by an increase in aromatase activity in asubject using the method as hereinbefore described, comprising comparingthe activity of aromatase in a sample taken from a first subject withthe activity in a sample taken from a second healthy control subject,wherein any increase in activity measured in the sample taken from thefirst subject relative to the second healthy control subject isindicative of disease.

In a seventh aspect of the present invention, there is provided a kitcomprising:

-   i) a compound as hereinbefore described; and-   ii) an assay buffer; and optionally-   iii) a stop buffer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the biochemical activity of aromatase in convertingandrostenedione to oestrone.

FIG. 2 shows a comparison of buffer only, control and CYP19 microsomeson fluorescence assay signal.

FIG. 3 depicts the effect that microsome volume has on fluorescenceassay signal.

FIG. 4 illustrates the NADPH dependence of the microsome preparation(CYP19)/aromatase enzyme activity.

FIG. 5 shows the specificity of the aromatase enzyme for its substrate.

DETAILED DESCRIPTION OF THE INVENTION Synthesis of Aromatase Substratei) Tert-Butyl2-{[(3-oxoandrost-4-en-17-yl)carbonyl]amino}-ethylcarbamate

To 0.49 g of 4-androsten-3-one-17β-carboxylic acid was addedN,N-dimethylformamide (3 ml), N,N-diisopropylethylamine (0.55 ml) andO—(N-Succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (0.48g). On stirring at room temperature (under an atmosphere of nitrogen)for 1.5 hours tert Butyl N-(2-aminoethyl)carbamate (0.25 g) was added.The mixture was stirred at room temperature for 3 days after which timethe volatile components were removed on a rotary evaporator. Flashcolumn chromatography was performed and the relevant fractions combinedand stripped of solvent using a rotary evaporator. This gave 0.50 g ofthe desired material (Formula XIII).

Mass spectrum: 459.30 (M+H)

ii) N-(2-aminoethyl)-3-oxoandrost-4-ene-17-carboxamide

To 16.5 mg of Tert-Butyl2-{[(3-oxoandrost-4-en-17-yl)carbonyl]amino}-ethylcarbamate was added0.5 ml of 95% trifluoroacacetic acid/water. On standing for 2 hours thevolatile components were removed using a rotary evaporator. Theresulting product (Formula XIV), which was an oil, was used withoutfurther purification. Mass spectrum: 359.23 (M+H)

iii)N-(2-{[2-(6,7,8,9,10-tetrahydro-14-sulfonato-16,16,18,18-tetramethyl-7aH-bisindolinium[3,2-a,3′,2′-a]pyrano[3,2-c,5,6-c]dipyridin-5-ium)acetyl]amino}ethyl)-3-oxoandrost-4-ene-17-carboxamide

To6,7,8,9,10-tetrahydro-2-carboxymethyl-14-sulfonato-16,16,18,18-tetramethyl-7aH-bisindolinium[3,2-a,3′,2′-a]pyrano[3,2-c,5,6-c]dipyridin-5-iumNHS ester (1.0 mg) was addedN-(2-aminoethyl)-3-oxoandrost-4-ene-17-carboxamide (0.6 mg),diisopropylethylamine (0.01 ml) and dichloromethane (0.2 ml). Thismixture was placed on a roller for 18 hours and then purified bypreparatory HPLC [column: Phenomenex JUPITER™ 10u C18 300A 250×21.2 mm.Method: 20 ml/min, 5% to 50% B over 30 min (A=water 0.1% TFA, B=CH3CN0.1% TFA). Peaks were detected at 559 nm. RT (product) ˜27 min].Relevant fractions were combined and concentrated on a rotaryevaporator. The material was then freeze dried to give 1.0 mg of thedesired product (Formula XV). Mass spectrum: 902 (M+H)

iv) Ethyl 6-(9-oxoacridin-10(9H)-yl)hexanoate

To 9(10H)-acridone (1.0 g) was added tetrahydrofuran (15 ml) under anatmosphere of nitrogen. Sodium hydride (0.25 g) was added with stirring;after 30 minutes ethyl 6-bromohexanoate (1.12 ml) was added and themixture heated to reflux for 18 hours. After this time water (10 ml) wasadded and the layers separated. The organic layer was dried overmagnesium sulfate, filtered and evaporated to dryness. Dry flash columnchromatography was performed to give 0.85 g of the required material(Formula XVI). Mass spectrum: 338 (M+H).

v) 6-(9-oxoacridin-10(9H)-yl)hexanoic acid

To ethyl 6-(9-oxoacridin-10(9H)-yl)hexanoate (0.80 g) was added aceticacid (9 ml) and 2M hydrochloric acid (2.5 ml). The mixture was heated to100° C. for 18 hours after which time the volatile components wereremoved on a rotary evaporator. Diethyl ether was added (25 ml) and themixture stirred for 15 minutes. The resulting material was filtered offand air dried to give 0.36 g final product (Formula XVII). Massspectrum: 310 (M+H).

vi) Tert-Butyl2-{[6-(9-oxoacridin-10(9H)-yl)hexanoyl]amino}ethylcarbamate

To 0.48 g of 6-(9-oxoacridin-10(9H)-yl)hexanoic acid was addeddichloromethane (6 ml) and thionyl chloride (0.2 ml). This mixture washeated to reflux for 1 hour after which time the volatile componentswere removed by application of vacuum. To the resulting oil was addeddichloromethane (3 ml), pyridine (3 ml) and t-butylN-(2-aminoethyl)carbamate (250 mg). This mixture was stirred for 18hours after which time it was poured into 0.5M sodium hydroxide solution(15 ml) and extracted with dichloromethane (2×10 ml). The combineddichloromethane solutions were washed with 0.1M hydrochloric acidsolution, dried over magnsium sulfate, filtered and evaporated todryness. The resulting material was purified by column chromatography togive 0.30 g of a solid final product (Formula XVIII).

vii) N-(2-aminoethyl)-6-(9-oxoacridin-10(9H)-yl)hexanamide hydrochloride

To 0.30 g of tert-butyl2-{[6-(9-oxoacridin-10(9H)-yl)hexanoyl]amino}ethylcarbamate was addeddicholoromethane (DCM; 30 ml). HCl (g) was bubbled through the solutionfor 10 minutes. After this time the mixture was filtered and washed withDCM (3×20 ml) to give the desired product (0.15 g; Formula XIX). Massspectrum: 352 (M+H).

viii) 3-oxo-N-(2-{[6-(9-oxoacridin-10(9H)-yl)hexanoyl]amino}ethyl)androst-4-ene-17-carboxamide

To 0.082 g of 4-androsten-3-one-17B carboxylic acid was added DMF (5ml), DIPEA (0.045 ml) andO—(N-Succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (0.08g). After stirring for 1 hourN-(2-aminoethyl)-6-(9-oxoacridin-10(9H)-yl)hexanamide hydrochloride(compound XVII) (0.10 g) was added and stirring continued for 3 days.Preparatory HPLC was performed [column: Phenomenex JUPITER™ 10u C18 300A250×21.2 mm; 20 ml/min, 5% to 95% B over 30 min (A=water 0.1% TFA,B═CH3CN 0.1% TFA). Peaks were detected at 280 nm. RT (product) ˜23 min]and the relevant fractions combined and concentrated on a rotaryevaporator. Freeze drying gave 0.0683 g of product (Formula XX). Massspectrum: 650 (M+H).

ix)5-ethyl-7,14-dioxo-12-{6-oxo-6-[(2-{[(3-oxoandrost-4-en-17-yl)carbonyl]amino}ethyl)amino]hexyl}-5,7,12,14-tetrahydroquino[2,3-b]acridin-2,9-disulfonicacid

N-(2-aminoethyl)-3-oxoandrost-4-ene-17-carboxamide (1.0 mg) wasdissolved in dichloromethane (1 ml) and a solution of5-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-12-ethyl-7,14-dioxo-5,7,12,14-tetrahydroquino[2,3-b]acridin-2,9-disulfonicacid (2 mg) in DMF (1 ml) added. DIPEA (0.02 ml) was added and themixture stirred at room temperature for 1 hour. After this timepreparatory HPLC was performed to give 1.6 mg of the desired material(Formula XXI). Mass spectrum: 956 (M+H).

Aromatase Assay

NADPH was prepared to a final concentration of 1 mM in 100 mM disodiumhydrogen phosphate buffer pH7.4. The labelled substrate (i.e.3-oxo-N-(2-{[6-(9-oxoacridin-10(9H)-yl)hexanoyl]amino}ethyl)androst-4-ene-17-carboxamide;Formula XIX) or chromophore alone (6-(9-oxoacridin-10(9H)-yl)hexanoicacid—compound XVI) was added to the solution of NADPH to a finalconcentration of 2 μM.

100 μl of this reagent was dispensed in to replicate wells of a 96 wellmicrotitre plate. To each well was dispensed 20 μ-30 μl of either theCYP19 (aromatase) containing microsomes or control microsomes (noCYP19). All microsomes were adjusted to the same protein concentrationwith assay buffer. The plates were incubated at 37° C. for 1 hour andthen fluorescence intensity measurements were recorded on the EnvisionPlate Reader (Perkin Elmer, US), excitation 405 nm/emission BFP450 nm.

FIG. 2 compares the fluorescence intensity data from a ‘buffer only’treatement and microsomes with and without aromatase activity. As can beseen, microsomes containing active aromatase produce a greater decreasein fluorescence intensity compared to the corresponding controlmicrosome preparation. The decrease in signal in the presence ofmicrosomes may represent quenching of the substrate signal due to thepresence of protein/lipid.

The fluorescence signal was seen to be proportional to the amount ofenzyme/microsome present in the assay. FIG. 3 depicts the effect ofmicrosome volume on the assay signal. A microsome volume of 20 μlgenerated a 17.5% decrease in intensity relative to the controlreaction. This was further increased to 24.2% in the presence of 30 μlof microsome preparation.

FIG. 4 illustrates the NADPH dependence of aromatase (CYP19 in thediagram) activity. A 24% decrease in fluorescence was observed in thepresence of additional NADPH. This compared to 16% in the absence ofadditional co-factor. The signal observed in the absence of additionalNADPH may reflect the presence of NAD(P)H in the enzyme preparation.

FIG. 5 shows the specificity of the enzyme for its substrate. In thepresence of 20 μl of CYP19 aromatase preparation a 17.5% change inintensity relative to the control was observed for the labelled steroidreporter. The chromophore alone (i.e. 6-(9-oxoacridin-10(9H)-yl)hexanoicacid—compound XVII) generated a 5% change in intensity when incubatedwith CYP19 microsomes. Therefore, the observed decrease in fluorescenceintensity was not due to the enzyme acting directly on the chromophore.

It is apparent that many modifications and variations of the inventionas hereinabove set forth may be made without departing from the spiritand scope thereof. The specific embodiments described are given by wayof example only, and the invention is limited only by the terms of theappended claims.

1. A compound of Formula 1:R-L-S   (I) wherein R is a fluorescent dye molecule; L is an optionallinkage group containing one or more atoms comprising hydrocarbon chainswhich may also contain other atoms such as N, O and S; and S is moleculecomprising a substrate group of the enzyme aromatase characterised inthat the fluorescence signal of said compound changes in respect offluorescence intensity or fluorescence lifetime when the compound isacted upon by an enzyme with aromatase activity.
 2. A compound accordingto claim 1 wherein R is selected from the group consisting offluorescein, rhodamine, coumarin, BODIPY™ dye, phenoxazine, cyanine,Alexa™ fluors, merocyanine, Cy3B, Cy5, Cy5.5, Cy7, acridone,quinacridone and squarate dyes
 3. A compound according to claim 1 or 2wherein said R is an acridone dye of Formula II:

wherein: groups R² and R³ are attached to the Z¹ ring structure andgroups R⁴ and R⁵ are attached to the Z² ring structure; Z¹ and Z²independently represent the atoms necessary to complete one or two fusedring aromatic or heteroaromatic systems, each ring having five or sixatoms selected from carbon atoms and optionally no more than two atomsselected from oxygen, nitrogen and sulphur; R¹, R², R³, R⁴ and R⁵ areindependently selected from hydrogen, halogen, amide, hydroxyl, cyano,amino, mono- or di-C₁-C₄ alkyl-substituted amino, sulphydryl, carbonyl,C₁-C₆ alkoxy, aryl, heteroaryl, C₁-C₂₀ alkyl, aralkyl; the group -E-Fwhere E is a spacer group having a chain from 1-60 atoms selected fromthe group consisting of carbon, nitrogen, oxygen, sulphur and phosphorusatoms and F is a target bonding group; and the group —(CH₂—)_(n)Y whereY is selected from sulphonate, sulphate, phosphonate, phosphate,quaternary ammonium and carboxyl and n is zero or an integer from 1 to6.
 4. A compound according to claim 1 or 2 wherein R is a quinacridonedye of Formula III:

wherein: groups R³ and R⁴ are attached to the Z¹ ring structure andgroups R⁵ and R⁶ are attached to the Z² ring structure; Z¹ and Z²independently represent the atoms necessary to complete one or two fusedring aromatic or heteroaromatic systems, each ring having five or sixatoms selected from carbon atoms and optionally no more than two atomsselected from oxygen, nitrogen and sulphur; R¹, R², R³, R⁴, R⁵, R⁶, R⁷and R⁸ are independently selected from hydrogen, halogen, amide,hydroxyl, cyano, amino, mono- or di-C₁-C₄ alkyl-substituted amino,sulphydryl, carbonyl, carboxyl, C₁-C₆ alkoxy, aryl, heteroaryl, C₁-C₂₀alkyl, aralkyl; the group -E-F where E is a spacer group having a chainfrom 1-60 atoms selected from the group consisting of carbon, nitrogen,oxygen, sulphur and phosphorus atoms and F is a target bonding group;and the group —(CH₂—)_(n)Y where Y is selected from sulphonate,sulphate, phosphonate, phosphate, quaternary ammonium and carboxyl and nis zero or an integer from 1 to
 6. 5. A compound according to any ofclaims 1 to 4 wherein L is a linker group containing from 1 to 40 linkedatoms selected from carbon atoms which may optionally include one ormore groups selected from —NR′—, —O—, —S—, —CH═CH—, —C≡C—, —CONH— andphenylenyl groups, wherein R′ is selected from hydrogen and C1 to C4alkyl.
 6. A compound according to any of claims 1 to 5, wherein L is alinker group containing from 2 to 30 atoms.
 7. A compound according toany of claims 1 to 6, wherein L is a linker group containing from 6 to20 atoms.
 8. A compound according to any of claims 1 to 7, wherein L isa linker group selected from the group:{(—CHR′—)_(p)-Q-(—CHR′—)_(r)}_(s) where each Q is selected from CHR′,NR′, O, —CH═CH—, Ar and —CONH—; each R′ is independently hydrogen or C₁to C₄ alkyl; each p is independently 0 to 5; each r is independently 0to 5; and s is either 1 or
 2. 9. A compound according to claim 8,wherein Q is selected from the group consisting of —CHR′—, —O— and—CONH—, where R′ is hydrogen or C₁ to C₄ alkyl.
 10. A compound accordingto any preceding claim wherein S is a substrate group of the enzymearomatase of formula IX

wherein: R¹ and R² are selected from H and methyl; R³ is selected fromH, C₁-C₈ alkyl, cyano, —(CH₂)_(k)—OR^(a); —(CH₂)_(k)—COOR^(a);—(CH₂)_(k)—SO₃R^(a); —(CH₂)_(k)—CHO, —(CH₂)_(k)—NR^(b)R^(c) and—(CH₂)_(k)—COR^(d); R⁴ is selected from H, —COR^(a) and hydroxyl; R⁵ isselected from H, —COR^(a), hydroxyl, cyano and halide; R⁶ is selectedfrom H and hydroxyl; R⁷, R⁸ and R⁹ are independently selected from H,—COR^(a) and hydroxyl; R¹⁰ is selected from H and halide; and whereR^(a) is selected from H and C1-C4 alkyl, optionally substituted withOH; R^(b) and R^(c) are selected from H and C₁-C₄ alkyl; R^(d) isselected from C₁-C₈ alkyl or C₁-C₈ alkyl optionally substituted withCOOR^(a), OH, OR^(a) or SO₃R^(a); and k is zero or an integer from 1 to8.
 11. A compound according to claim 10 wherein Group S is a steroidselected from the group of steroid families consisting of4-androsten-3-one, 4-cholesten-3-one, 4-estren-3-one and 4-pregnen-3-onederivatives.
 12. A compound according to any of claims 1 to 11 wherein Sis androstenedione of Formula X or a derivative thereof.


13. A compound according to any of claims 1 to 11 wherein S istestosterone of Formula XI or a derivative thereof.


14. A compound according to any preceding claim of Formula XX


15. A method for measuring aromatase activity in a sample, the methodcomprising the steps of: i) measuring the fluorescence intensity orfluorescence lifetime of a compound according to any preceding claimprior to adding it to said sample; ii) adding said compound to saidsample under conditions which favour aromatase activity, and iii)measuring a change in fluorescence intensity or fluorescence lifetime ofsaid compound following step ii); wherein said change in fluorescenceintensity or fluorescence lifetime can be used to determine aromataseactivity.
 16. A method according to claim 15 wherein the sample isselected from the group consisting of extract, cell, tissue andorganism.
 17. A method of screening for a test agent whose effect uponthe activity of aromatase is to be determined, said method comprisingthe steps of: i) performing the method of claim 15 or 16 in the presenceof said agent; and ii) comparing the activity of said aromatase in thepresence of the agent with a known value for the activity of aromatasein the absence of the agent; wherein a difference between the activityof the aromatase in the presence of the agent and said known value inthe absence of the agent is indicative of the effect of the test agentupon the activity of aromatase.
 18. The method according to claim 16,wherein the known value is stored upon an electronic database.
 19. Amethod of screening for a test agent whose effect upon the activity ofaromatase is to be determined, said method comprising the steps of: i)performing the method of claim 16 or 17 in the presence and in theabsence of the agent; and ii) determining the activity of said enzyme inthe presence and in the absence of the agent; wherein a differencebetween the activity of aromatase in the presence and in the absence ofthe agent is indicative of the effect of the test agent upon theactivity of aromatase.
 20. The method according to claim 18 wherein saiddifference in activity between the activity of aromatase in the absenceand in the presence of the agent is normalised, stored electronicallyand compared with a value of a reference compound.
 21. A method formeasuring the distribution of a compound of any of claims 1 to 14 withina tissue, wherein the compound is capable of being taken up by a livingcell within said tissue, the method comprising the steps of: i)measuring the fluorescence intensity or fluorescence lifetime of thecompound in a cell-free environment or a parental host cell; ii) addingthe compound to one or more cells or a cell engineered to over-expressaromatase, and iii) measuring the fluorescence intensity or lifetime ofthe compound following step ii); wherein a change in fluorescenceintensity or fluorescence lifetime indicates aromatase activity and canbe used to determine the distribution of the compound.
 22. A methodaccording to claim 21, wherein the distribution of the compound withinthe tissue of a first subject is compared with the distribution of thecompound within the tissue of a second subject.
 23. The method of claim22, wherein said subject is selected from the group consisting ofmammal, plant, insect, fish, bird, fly, nematode and algae.
 24. Themethod of claim 23, wherein the mammal is a mouse or a rat.
 25. Use of acompound according to any of claims 1 to 14 for measuring aromataseactivity as an in vitro or an in vivo imaging probe.
 26. A method ofdiagnosing a disease caused by an increase in aromatase activity in asubject using the method according to claim 15, comprising comparing theactivity of aromatase in a sample taken from a first subject with theactivity in a sample taken from a second healthy control subject,wherein any increase in activity measured in the sample taken from thefirst subject relative to the second healthy control subject isindicative of disease.
 27. Kit comprising: i) a compound according toany of claims 1 to 14; ii) an assay buffer; and optionally iii) a stopbuffer.